An endogenous factor or family of factors found in animal and human tissue and fluids has been associated with natriuresis, the excretion of abnormal amounts of sodium in the urine. The natriuretic action is believed to result from the inhibition of the Na,K-ATPase enzyme system which mediates active transepithelial sodium transport. Studies have also suggested that this factor may play a pathological role in hypertension. See S. Graves et al., "An Endogenous Digoxin-Like Substance in Patients with Renal Impairment," Annals of Internal Medicine, 99: 604-608 (1983); M.-A. Devynck et al., "Measurement of Digitalis-Like Compound in Plasma: Application in Studies of Essential Hypertension," British Medical Journal, 287: 631-634 (1983); and R. Valdes et al., "Endogenous Substance in Newborn Infants Causing False Positive Digoxin Measurements," Journal of Pediatrics, 102: 947-950 (1983). An association between serum levels of this factor and hypertension in pregnancy has also been found. S. Graves et al., "Endogenous Digoxin--Immunoreactive Substance in Human Pregnancies," J. Clinical Endocrinol. and Metab., 58:748 (1984).
This factor or family of factors cross-reacts with antibodies raised against digoxin, a drug of the cardiac glycoside family. R. Valdes et al., "Protein Binding of Endogenous Digoxin-Immunoactive Factors in Human Serum and Its Variation with Clinical Condition," J. of Clinical Endocrinol. and Metab., 60: 1135-1143 (1985).
As used for purposes of the present invention, this factor is referred to as a digitalis-like factor. It will be understood by those of skill in the art, that other terms may have been used to describe this factor, including digoxin-like and ouabain-like.
Digitalis, digoxin, and ouabain are chemically and pharmacologically related cardiac glycosides, which are used to treat patients with cardiac malfunctions. By increasing the strength of heart muscle contraction, the drugs are indicated in the treatment of congestive heart failure, in atrial flutter and fibrillation, and in paroxysmal atrial tachycardia. There are a significant number of cardiac patients with impaired renal function that are treated with cardiac glycosides. Moreover, patients with renal impairment or renal failure often develop congestive heart failure or malfunctions that are then treated with cardiac glycosides. Remington's Pharmaceutical Sciences (16th Ed. 1980) at 654-655, 794-798.
The presence of this digitalis-like factor in serum has a detrimental impact on the measurements of cardiac glycosides by present techniques. Studies have shown that this endogenously produced factor measures as digoxin in the serum of digoxin-free patients with renal impairment. S. Graves et al., "An Endogenous Digoxin-Like Substance in Patients with Renal Impairment," Annals of Internal Medicine, 99: 604-608 (1983).
Although the existence of this factor is known, it has yet to be substantially purified and identified. See E. Clarkson et al., "Further Observations on a Low-Molecular-Weight Natriuretic Substance in the Urine of Normal Man," Kidney International, 16: 710-721 (1979). The Clarkson study describes a partial purification of the factor from the urine of a normal man, suggesting that the factor may have a molecular weight below 500 daltons; that the natriuretic activity of the factor is relatively resistant to heat at high or low pH, and to oxidation with nitrous acid; that it is insoluble in the less polar organic solvents; and that it is destroyed by prolidase. The factor was separated from the urine using a chromatography column of G-25 Sephadex. The authors suggested that the natriuretic activity is due to a peptide containing the heterocyclic amino acid, proline.
In H. Kramer et al., "Further Studies on Isolation and Purification of a Small Molecular Weight Natriuretic Hormone," Hormonal Regulation of Sodium Excretion (1980), 303-323, the authors describe studies on the isolation and partial purification of the factor from human urine. Urine is treated by such steps as gel filtration, high pressure liquid chromatography, reverse phase chromatography, ion exchange chromatography and electrophoresis. At page 320, the authors suggest that the factor may be a small acidic peptide.
J.-F. Cloix et al., "Purification of an Endogenous Inhibitor of Sodium-Potassium-ATPase," Endocrinologie, C. R. Acad. Sc. Paris, 296: 213-216 (1983), described the partial purification of the factor from human plasma using gel filtration, followed by anion exchange and high pressure liquid chromatography on reverse phase. M. Crabos et al., "Measurement of Endogenous Na.sup.+, K.sup.+ -ATPase Inhibitors in Human Plasma and Urine Using High-Performance Liquid Chromatography," FEBS Letters, 176: 223-228 (1984), used reverse-phase HPLC coupled with Na, K-ATPase inhibition and cross-reaction antibodies to isolate the factor from human plasma and urine. I. W. Wainer, "Rapid Large-Scale Isolation of Biologically Active Molecules Using Reversed-Phase `Flash` Chromatography: Initial Purification of Endogenous Na.sup.+, K.sup.+ -ATPase Inhibitors from Human Urine," Journal of Chromatography, 338: 417-421 (1985), used the method of M. Crabos et al. using "flash" chromatography. J.-F. Cloix et al., "Purification from Human Plasma of Endogenous Sodium Transport Inhibitor(s)," Experientia, 40: 1380-1382 (1984), used a purification process for the factor from human plasma comprising (a) inhibition of Na, K-ATPase activity; (b) inhibition of .sup.3 H-ouabain binding; and (c) cross-reactivity with antidigoxin antibodies. H. de The et al., "Plasma Sodium Pump Inhibitor in Essential Hypertension and Normotensive Subjects with Hypertensive Heredity," Journal of Cardiovascular Pharmacology, 6: 549-554 (1984), described isolating the factor from human plasma by Na, K-ATPase activity and by ouabain binding inhibition. M-A. Devynck et al., "Circulating Digitalis-Like Compounds in Essential Hypertension," Clin. and Exper. Hyper.--Theory and Practice, A6: 441-453 (1984), isolated a partially purified factor by gel filtration, anion exchange chromatography, and HPLC on reverse phase.
Applicant herein has recognized one of the major problems of isolating and purifying the digitalis-like factor: the vast majority of the factor is protein bound and thus must be separated from its protein binding sites in order to be purified. The seeming failure of the researchers in this area to recognize this problem has limited their success to the partial purification of the factor described above. Indeed, Applicant is the first to isolate and purify the digitalis-like factor to the degree of purity shown in the present invention.
One of the attendant advantages of Applicant's discovery is that it provides for a process which yields sufficient digitalis-like factor (DLF) to permit the factor to be physically and chemically identified. A major difficulty previously encountered in isolating the factor was insufficient quantities of the material. With sufficient yield of the factor, it can be purified and identified. More importantly, once the factor is identified, its potential role as cause or mediator of essential hypertension or pregnancy-induced hypertension can be readily assessed. Such a hypertensionogenic factor has clinical utility and can be used to produce antibodies for passive immunity, to produce immunogenic forms for active immunity, and to produce analogues that can act as antagonists. Any of these uses can then lead to different therapeutic modalities to reduce blood pressure.
The factor, by increasing vascular resistance, also has a therapeutic role in the acute treatment of hypotensive states such as shock. Additionally, because the factor cross-reacts with digoxin antibodies and can inhibit the ouabain-sensitive Na,K-ATPase analogous to digoxin, the factor has utility as an endogenous cardiotropic (ionotropic) agent capable of modifying cardiac function, and can be used as a therapeutic agent in the treatment of congestive heart failure or cardiac arrhythmias, currently treated with cardiac glycosides.